Biochemical and biological roles of ribosome-inactivating protein

Abstract

Ribosome-inactivating proteins (RIPs) are a group of plant cytotoxic N-glycosidases that specifically cleave nucleotide N-C glycosidic bonds. It has been proposed that RIPs inhibit protein synthesis by virtue of their enzymatic activity, selectively removing a specific adenine residue from the highly conserved and surface-exposed α-sarcin/ricin (S/R) loop in the large rRNA. This enzymatic cleavage prevents the binding of the EF-2/GTP complex to the ribosome, with the subsequent arrest of protein synthesis, and, eventually, cell death. In the present study, a novel type-1 RIP, termed PAP-H, was purified from Agrobacterium rhizogenes-transformed hairy roots of Phytolacca americana, and biochemically characterized. PAP-H was determined to be 29.5 kD, and its full-length cDNA sequence has 1,125 nucleotides with an open reading frame of 1074 nucleotides representing 339 amino acids (Genebank, accession number AY071928). Further, PAP-H was found to be localized in the cell walls of hairy roots and root border cells using immuno-fluorescence microscopy, and was determined to be constitutively secreted as part of the root exudates, with its secretion enhanced by a mechanism mediated by ethylene induction. By secretion into the rhizosphere, PAP-H penetrates and inhibits the growth of soil-borne fungi by a synergetic combination of chitinase, β-1,3-glucanase, and protease within root exudates. To further understand the antifungal mechanism of RIPs, I have examined three type I RIPs to determine their substrate specificities on fungal ribosomes, and I have correlated the data with their antifungal activity. The results provide experimental evidence that the enzymatic activity and specificity of RIPs are separated from their cytotoxicity. In addition, using protein-fluorescence labeling technology, I have found that RIPs selectively recognize, interact with, and are internalized into fungal cells to exert cytotoxicity. The final stage of this research focused on investigating the enzymatic interaction of the RIP ME1 (from Mirabilis expansa) with non-ribosomal substrates upon their structural modification. The RIP-RNA/ RIP-DNA interactions was found to depend on the secondary structure of substrates, and did not require any sequence or structure motif. RIP targeted only single-stranded regions of nucleic acid, removing adenine and, to a lesser extent, guanine residues. In addition, RIP was capable of recognizing and depurinating a pathogenic RNA transcript, completely abolishing its infectivity. Subsequently, we examined the activity of ME1 toward M. expansa ribosomes in the presence of increasing concentrations of small synthetic oligonucleotides (23SS0-A12), indicating that the long-considered only substrate of RIP, the ribosome, is unlikely be the primary target in plant systems.